植物内生菌活性代谢产物最新研究进展

内生菌广泛存在于植物组织中,作为一种新型的微生物资源,具有丰富的物种多样性,也是发掘新型天然活性物质的重要途径之一,有些内生菌还能产生与宿主植物相同或相似的活性成分。因此从植物内生菌中挖掘抗菌尤其抗临床耐药菌、抗肿瘤等天然活性产物不仅为新药的研发提供了新的方向,还能在一定程度上解决传统的天然产物药源——药用植物生长缓慢、资源紧缺等问题。从多个角度概述了近年来国内外报道的植物内生菌次生代谢产物及其来源、生物活性等方面的主要成果和最新进展,以期为植物内生菌活性代谢产物的研究提供参考。     

基于植物内生菌及其次生代谢活性物质多样性的研究

据相关研究表明,植物内生菌属于新型微生物资源,并且种类较多,分布广泛,譬如在最近几年,科学家逐渐发现在植物内生菌中发现了紫杉醇等生物活性物质以及在一些产物中还发现了与次级代谢产物中相同的产物,因此对临床研究具有重要价值和潜在价值。对此研究的主题为:基于植物内生菌及其次生代谢活性物质多样性的研究。本为将从植物内生菌的多样性,植物内生菌次生代谢活性物质的多样性等方面进行逐一研究,现将文章综述如下。     

药用植物内生真菌研究进展

药用植物内生真菌具有抗肿瘤、抗菌、抗病毒、抗氧化等活性,能够产生植物生长素、细胞激动素、赤霉素等促植物生长物质促进植物生长,从而使研究药用植物内生真菌成为寻找新型拮抗菌、抗肿瘤、抗氧化活性药物的重要资源。本文对药用植物内生真菌的多样性、分离鉴定、产生的次生代谢产物及其功能等做一综述。     

植物内生放线菌多样性研究进展

植物内生放线菌作为植物内生菌资源的一大类,具有丰富的多样性,其次级代谢产物也十分丰富,而且还具有各种生物学活性。通过了解植物内生放线菌资源的多样性,可以对其产生的生物活性物质进行筛选和分析,从而筛选出具有良好药理活性的物质应用于临床。从植物内生放线菌宿主植物、组织分布、种类和数量以及活性基因4个方面综述了植物内生放线菌的多样性资源,介绍植物内生放线菌新的微生物物种,总结了植物内生放线菌目前研究中存在的问题及展望。     

剑叶龙血树内生真菌的分离及体外抑菌活性筛选

为研究剑叶龙血树内生真菌资源多样性,初步探讨和筛选具有抑菌活性的特异性菌株以及进一步开发剑叶龙血树内生真菌的抗菌活性化合物。该文采用植物组织分离法从剑叶龙血树茎和叶中分离内生真菌,对内生真菌进行液体发酵7 d,经乙酸乙酯萃取后制得粗提物,并采用牛津杯扩散法,以10种常见病原菌和5种临床耐药菌为靶标检测其发酵粗提物的抑菌活性,对有较好抑菌活性的内生真菌进行分子鉴定。结果表明:(1)从剑叶龙血树茎、叶中共分离得到345株内生真菌,294株对一种以上指示菌有抑制活性;(2)其中84株内生真菌对5株临床耐药菌均有不同程度的抑制活性,占所分离菌株总数的24.35%,75%的内生真菌对金黄色葡萄球菌有抑制活性。这说明剑叶龙血树中存在多种有抑菌活性的内生真菌,为剑叶龙血树内生菌抗菌活性成分挖掘及新型抗菌药物筛选奠定了基础。     

蜘蛛抱蛋属植物内生细菌的多样性及其抗菌活性筛选

【背景】药用植物中蕴含多样性丰富的内生菌资源,这些微生物产生的多种新型物质在制药领域表现出较好的应用前景。【目的】研究蜘蛛抱蛋属(Aspidistra Ker-Gawl.)植物内生细菌的多样性,探索药用植物内生细菌在药用活性产物方面的开发潜力,以期发现具有抗菌活性的次级代谢产物。【方法】对9种13株新鲜的蜘蛛抱蛋植物进行表面消毒,采用5种分离培养基分离内生细菌;根据菌落形态特征排除重复菌株,并测定其16S rRNA基因序列,构建系统进化树分析内生细菌多样性;将菌株分别用2种培养基发酵,使用耻垢分枝杆菌(Mycobacterium smegmatis ATCC 700044)、水稻白叶枯菌(Xanthomonas oryzae PXO99A)、白色念珠菌(Candidaalbicans ATCC 10231)、肺炎雷伯菌(Klebsiella pneumoniae ATCC 700603)和耐药粪肠球菌HH22(Enterococcus faecalis HH22)5种检定菌对分离菌株的发酵液进行抑菌活性筛选。【结果】从植物组织中分离得到了234株内生细菌,根据形态初步排重得到156株植物内生细菌;基于16S rRNA基因序列构建的系统进化树显示它们分属于3门10目22科29属,其中链霉菌属(Streptomyces)、芽孢杆菌属(Bacillus)、微杆菌属(Microbacterium)、类芽孢杆菌属(Paenibacillus)和根瘤菌属(Rhizobium)的菌株广泛地分布在不同种的蜘蛛抱蛋植株中,且占据一定优势;发现可能的潜在新分类单元6个;156株内生细菌中38株菌的发酵液具有抑菌活性,初筛阳性率为23.7%。【结论】蜘蛛抱蛋植物组织中含有种类多样的内生细菌,它们可能是抗菌生物活性次级代谢产物的有效来源。     

云南重楼植物内生真菌的分离及抗菌活性筛选

从云南重楼[Paris polyphylla Smithvar.yunnnanensis(Franch.)Hand.Mazz.]块状茎中分离出166株内生真菌,对其进行形态分类鉴定归于4目,6科,20个属,体现了云南重楼植物内生真菌的生物多样性特征。同时,选择与人类和植物相关的37株病原微生物作为抗菌活性筛选指示菌,进行了云南重楼植物内生真菌抗菌活性的初步研究。结果表明,4株内生真菌对细菌、植物致病真菌、皮肤致病真菌多种病原微生物具有显著抑制生长的作用。     

川乌植物内生真菌抗菌活性的研究

从川乌（Radix aconiti）植物的根和茎中分离获得内生真菌61株,经显微形态观察进行内生真菌的分类鉴定,同时采用纸片法进行抗菌活性筛选。结果有29株内生真菌显示抗菌活性,占菌株数的47．54％;毛壳菌属是抗菌活性菌株的优势种属;活性高且抗菌谱广的菌株有3株,主要分布在毛壳菌属和束丝菌属。结果表明：川乌植物内生真菌中广泛分布着有抗菌活性的菌株。     

植物内生真菌及其活性代谢产物研究进展

植物内生真菌是一大类未被充分研究过的真菌,其物种及代谢产物均具有生物多样性,现今从植物内生真菌中得到的活性物质种类远比从土壤微生物中得到的多。对植物内生真菌的研究具有重要的生态学意义和经济学意义,在各个领域中应用前景广泛。作者主要综述了植物内生真菌及其活性物质研究的最新进展。     

北部湾徐闻海域红树内生细菌物种多样性及其杀线虫活性研究

为发掘红树植物内生细菌资源和寻找新型微生物杀虫剂,该研究从北部湾徐闻海域采集7种红树植物共16份样品,设计10种分离培养基,使用稀释涂布法分析红树植物内生细菌的分布特征,通过16S rRNA分子生物学方法对内生细菌进行多样性分析,并利用秀丽隐杆线虫模型,通过杀线虫活性实验测试内生细菌乙酸乙酯提取物的杀线虫活性。结果表明:(1)从16份红树植物各组织器官中获得33株内生细菌,分布于19个科23个属。其中,芽孢杆菌属(Bacillus)为优势菌属,并发现了10株潜在的新种或新属。(2)筛选到具有显著杀线虫活性的菌株IMDGX 4725和IMDGX 4744,半数致死浓度(LC_(50))分别为61.58、100.89mg·mL~(-1)。研究结果证实了徐闻海域红树具有多样性丰富的内生细菌,同时部分细菌具有较强的杀线虫活性,具有发现新型微生物杀虫剂的潜力。     

药用植物青蒿不同种类的内生菌抑菌活性分析

为了研究青蒿不同种类的内生菌抑制细菌和抑制真菌的活性,该研究采用组织块法和研磨法从青蒿的根、茎、叶中分离内生细菌、放线菌和真菌,以大肠埃希菌(Escherichia coli)(CICC 23657)、枯草芽孢杆菌(Bacillus subtilis)(CICC 10275)、金黄色葡萄球菌(Staphylococcus aureus)(CICC 10384)、黑曲霉(Aspergillus niger)(CICC 2487)、酿酒酵母(Saccharomyces cerevisiae)(CICC 33032)为指示菌,采用琼脂块法和双层平板法检测内生菌的抑菌活性。结果表明:(1)从青蒿植株中共分离到76株内生菌,其中内生细菌19株、内生放线菌34株、内生真菌23株。从分离部位来看,56株来自于茎段、17株来自于根段、3株来自叶片。(2)内生细菌中抑菌活性菌株占总菌株的比例最高,为95%,内生放线菌和内生真菌中抑菌活性菌株的比例分别为41%、35%。(3)内生细菌的抗菌谱较广;虽然内生放线菌的抗菌谱较窄,但其中高抗菌株较多,尤其对酿酒酵母的抑菌效果好。综上结果显示,药用植物青蒿中存在着丰富的有抑菌活性的内生菌,且不同种类的内生菌抑菌活性不同。     

Bioactive sesquiterpene,plasticizer, and phenols from the fungal endophytes of <Emphasis Type="Italic">Polygonum chinense</Emphasis> L.

There is a constant need for novel antibiotic and antioxidant sources due to the ever-increasing resilience of pathogens and the occurrence of chronic diseases. The natural sources of these agents have advantages due to lower production cost, structural variation, and uses of active compounds for pharmaceutical uses. The microbes living in planta termed “endophytes” are alternative sources of host bioactive compounds. In this study, ten endophytic fungi were isolated from Polygonum chinense L. and identified by sequencing of the internal transcribed spacer regions. The fungal strains were fermented and the ethyl acetate extracts were evaluated for antimicrobial and antioxidant capacities. Almost 80% of the endophytes showed antibacterial potency against one or more pathogenic bacteria. Among all strains, Penicillium canescens showed broad-spectrum antimicrobial activity against gram-positive and gram-negative pathogens as well as significant antioxidative and DNA protective capacities. The strain Fusarium chlamydosporum displayed significant anti-radical (126.8?±?6.7 μg/ml) and ferric reducing (84.7?±?2.1 mg AA/g dry extract) capacities. The bio-autography, chromatography, and mass spectroscopy analyses of P. canescens extract revealed the presence of sesquiterpene (germacrene), plasticizer (phthalic acid ester) along with phenolic acids, flavonoid (quercetin), and short chain hydrocarbons. The secondary metabolites of F. chlamydosporum were identified with phenolic acids as bioactive compounds by chromatography and mass spectroscopy. This study indicates P. chinense endophytes as potential sources of antimicrobial and antioxidant compounds for novel drug discovery.     

Antimicrobial fungal endophytes from the botanical medicine goldenseal (Hydrastis canadensis)

The potential of fungal endophytes to alter or contribute to plant chemistry and biology has been the topic of a great deal of recent interest. For plants that are used medicinally, it has been proposed that endophytes might play an important role in biological activity. With this study, we sought to identify antimicrobial fungal endophytes from the medicinal plant goldenseal (Hydrastis canadensis L., Ranunculaceae), a plant used in traditional medicine to treat infection. A total of 23 fungal cultures were obtained from surface-sterilized samples of H. canadensis roots, leaves and seeds. Eleven secondary metabolites were isolated from these fungal endophytes, five of which had reported antimicrobial activity. Hydrastis canadensis plant material was then analyzed for the presence of fungal metabolites using liquid chromatography coupled to high resolving power mass spectrometry. The antimicrobial compound alternariol monomethyl ether was detected both as a metabolite of the fungal endophyte Alternaria spp. isolated from H. canadensis seeds, and as a component of an extract from the H. canadensis seed material. Notably, fungi of the Alternaria genus were isolated from three separate accessions of H. canadensis plant material collected in a time period spanning 5 years. The concentration of alternariol monomethyl ether (991 mg/kg in dry seed material) was in a similar range to that previously reported for metabolites of ecologically important fungal endophytes. The seed extracts themselves, however, did not possess antimicrobial activity.     

Biogenic synthesis of gold nanoparticles by marine endophytic fungus-Cladosporium cladosporioides isolated from seaweed and evaluation of their antioxidant and antimicrobial properties

Marine endophytes are the most untapped group of microorganisms having enormous applications in pharmaceutical and cosmetra id="spar0060">Marine endophytes are the most untapped group of microorganisms having enormous applications in pharmaceutical and cosmetic industries. In the present study, we have optimized a method for biogenic synthesis of gold nanoparticles (AuNPs) from Cladosporium cladosporioides, an endophytic fungus of the seaweed, Sargassumwightii. The identity of the fungus was established by the 18 s rRNA and ITS sequence. The AuNPs synthesized using C. cladosporioides were characterized by UV–vis spectrophotometer, Field Emission Scanning Electron Microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, Dynamic light scattering, Atomic force microscopy, and Energy dispersive X-ray spectroscopic studies. They were tested for free radical scavenging activity (DPPH and FRAP assay) and antimicrobial activity against a panel of pathogenic microorganisms. The AuNps were within 100 nm as confirmed by the above methods. An attempt was made to understand the mechanism of the gold nanoparticle synthesis using the fungal extract. The present study shows the involvement of NADPH-dependent reductase and phenolic compounds in the bioreduction of the gold metal salts to nanoparticles. The AuNPs showed significant antioxidant as well as the antimicrobial activity. Hence, this study has shown a great potential for the development of a cost effective antimicrobial treatment utilizing biogenic gold nanoparticles.     

Comparison of the chemistry and diversity of endophytes isolated from wild-harvested and greenhouse-cultivated yerba mansa (Anemopsis californica)

With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 μg/mL, and three inhibited growth of Pseudomonas aeruginosa by ≥20% at 200 μg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional 14 known compounds were identified using LC–MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi.     

Naturally occurring bioactive Cyclobutane-containing (CBC) alkaloids in fungi,fungal endophytes,and plants

This article focuses on the occurrence and biological activities of cyclobutane-containing (CBC) alkaloids obtained from fungi, fungal endophytes, and plants. Naturally occurring CBC alkaloids are of particular interest because many of these compounds display important biological activities and possess antitumour, antibacterial, antimicrobial, antifungal, and immunosuppressive properties. Therefore, these compounds are of great interest in the fields of medicine, pharmacology, medicinal chemistry, and the pharmaceutical industry. Fermentation and production of CBC alkaloids by fungi and/or fungal endophytes is also discussed. This review presents the structures and describes the activities of 98 CBC alkaloids.     

药用植物内生菌的分离及抗菌活性的初步研究

为研究药用植物金荞麦、款冬及黄皮树中内生菌的抗菌活性,筛选具有广谱抗菌效果的内生菌。以导致人畜及植物致病的15种病原微生物为拮抗菌株,采用改进K-B纸片法、生长速率法及琼脂二倍稀释法进行抑菌试验。结果从三种药用植物中共分离到内生菌38株,其中FE-R9、TE-R7、PE-S11三株内生真菌发酵液提取物对10种人畜指示菌的最低抑菌浓度(M IC)分别在7.50~15.00 g.L-1、3.13~12.50 g.L-1、0.39~15.00 g.L-1之间。FE-R9、TE-R7、PE-S11对多种病原微生物具有显著抑制生长的作用,这可为寻找天然抗菌物质提供资源。     

Endophytic fungi as novel sources of biopesticides: the Macaronesian Laurel forest,a case study

Endophytes fungi have been widely bioprospected to find new drugs and drug leads including antimicrobial agents and antifungals. However, an important role in host plant protection has been suggested for their presence and their metabolites. Therefore, nematicidal and insecticidal effects of their metabolites should be expected. In this review, the literature data available on insecicidal and nematicidal compounds identified from fungal endophytes are presented. Additionally we present a recent study on the endophytic biodiversity of a unique paleoflora, the Macaronesian laurel forest, in the light of their role in plant protection.     

Endophytic Penicillium species secretes mycophenolic acid that inhibits the growth of phytopathogenic fungi

The worldwide demand for reduced and restricted use of pesticides in agriculture due to serious environmental effects, health risks and the development of pathogen resistance calls for the discovery of new bioactive compounds. In the medical field, antibiotic-resistant microorganisms have become a major threat to man, increasing mortality. Endophytes are endosymbiotic microorganisms that inhabit plant tissues without causing any visible damage to their host. Many endophytes secrete secondary metabolites with biological activity against a broad range of pathogens, making them potential candidates for novel drugs and alternative pesticides of natural origin. We isolated endophytes from wild plants in Israel, focusing on endophytes that secrete secondary metabolites with biological activity. We isolated 302 different endophytes from 30 different wild plants; 70 of them exhibited biological activity against phytopathogens. One biologically active fungal endophyte from the genus Penicillium, isolated from a squill (Urginea maritima) leaf, was further examined. Chloroform-based extraction of its growth medium was similarly active against phytopathogens. High-performance liquid chromatography separation followed by gas chromatography/mass spectrometry analysis revealed a single compound—mycophenolic acid—as the main contributor to the biological activity of the organic extract.     

Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea

An endophytic fungus displaying considerable antimicrobial activity was isolated from stem tissue of an invasive plant species, Ipomoea carnea. The fungus was identified as Quambalaria sp. and confirmed by ITS rDNA sequence analysis. A BLAST search result of the sequence indicated 97 % homology with Quambalaria cyanescens. Crude metabolites of the fungus showed considerable antimicrobial activity against a panel of clinically significant microorganisms. The metabolites showed highest in vitro activity against Shigella dysenteriae followed by Escherichia coli and Candida albicans. Optimum metabolites production required neutral pH and a 15-day incubation period. Bark extracts amended with fungal media demonstrated higher antimicrobial activity. Optimum metabolites activity was recorded in Czapek Dox broth amended with leaf extracts (CDB + LE) of the host plant. The metabolites showed UV λ-max in ethyl acetate at 284.6 nm with an absorbance value of 1.093. Phylogenetic tree generated by the Maximum Parsimony method showed clustering of our isolate with Q. cyanescens with supported bootstrap of 65 %. Species of Quambalaria are pathogens to Eucalyptus and occurrence of this fungus as endophytes support it to be a latent pathogen. Sequence base analysis and RNA secondary structure study also confirmed such a relationship. Secondary structural features like two hinges and a 5’ dangling end were found to be unique to our isolate. These structural features can also be used as potential barcodes for this fungus. The findings indicate that invasive plant species can be a reliable source of novel endophytes with rich antimicrobial metabolites. The study also validates the assumption that endophytes can become parasites and share a close affinity.